Attachment of second harmonic-active moiety to molecules for detection of molecules at interfaces

ABSTRACT

This invention provides methods of detecting molecules at an interface, which comprise labeling the molecules with a second harmonic-active moiety and detecting the labeled molecules at the interface using a surface selective technique. The invention also provides methods for detecting a molecule in a medium and for determining the orientation of a molecular species within a planar surface using a second harmonic-active moiety and a surface selective technique.

Government support under grant number CHE-96-12685 from the NationalScience Foundation and grant numbers DE-FG02-91ER and DE-FG02-14226 fromthe Department of Energy. Accordingly, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced inparentheses by author and year. Full citations for these references maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

Second harmonic generation (SHG) is a powerful spectroscopic tool forstudying interfacial regions at the molecular scale, but to date hasbeen confined mainly to non-biological systems. Recently, however, SHGhas been extended to the study of a SH-active moiety at a supportedlipid membrane system (Salafsky and Eisenthal, 2000a), a useful modelfor biological studies, and to the detection of protein adsorption atcharged interfaces through the indirect effect the protein has onpolarized water molecules near the surface (Salafsky and Eisenthal,2000b). Direct detection of molecules such as proteins at interfacescould be useful in a number of biological studies, for example instudies of protein-receptor binding at a membrane or cellular interfaceor in the development of biosensors, but is hindered by theintrinsically low SH cross-section of proteins. For detection ofmolecules by SHG, the SH-active moiety must possess ahyperpolarizability and a net orientation at the interface. Althoughsome proteins do contain chromophoric cofactors which are SH-active,their absorption is usually quite low or they are centrosymmetric. Othersources of SH activity in proteins include the aromatic amino acid sidechains which are weakly SH-active. However, their varying orientationswithin the protein would reduce any SH signal.

The present application discloses the concept and technique of a‘SHG-label’. SHG labels are second harmonic-active moieties which can beattached to a molecule or particle of interest that is not SH-active oronly weakly SH-active, in order to render the molecule amenable to studyat an interface. The labeled molecules may then be studied bysurface-selective techniques such as second harmonic generation orsum-frequency generation. The technique can be illustrated by covalentlylabeling a protein, cytochrome c, with a SH-active moiety which isspecific for either amine or sulfhydryl groups, common chemical moietieswhich exist on the surface of many protein molecules as part of theiramino acid side-chains. Unlike detection with fluorescent labels,SHG-labels have the important advantage that only labeled proteins at aninterface, and with a net orientation, contribute to the second harmonicsignal; labeled protein molecules in the bulk contribute no signal.Furthermore, unlabeled molecules at the interface are undetectable.SHG-labels should find use in a variety of biological applicationsincluding studies of protein-protein, protein-membrane, and cell-cellinteractions. SHG-labels can also be used to study other systems such asnanoparticle surfaces and polymer systems (polymer beads). In turn thelabelled nanoparticle or labelled polymer bead can be used for exampleas a sensor of molecules in the surrounding medium.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting a molecule at aninterface, which comprises labeling the molecule with a secondharmonic-active moiety and detecting the labeled molecule at theinterface using a surface selective technique.

The invention also provides a method for detecting a molecule in amedium, which comprises:

-   (a) labeling a surface with a second harmonic-active moiety wherein    the second harmonic-active moiety specifically interacts with the    molecule to be detected,-   (b) exposing the surface to the medium thereby creating an interface    at the surface,-   (c) detecting the second harmonic-active moiety at the interface by    measuring a signal generated using a surface selective technique,    and-   (d) detecting a change in the signal when the molecule interacts    with the second harmonic-active moiety, thereby detecting the    molecule in the medium.

This invention provides a method for determining the orientation of amolecular species within a planar surface, which comprises:

-   (a) labeling the species with a second harmonic-active moiety which    specifically binds to the species;-   (b) determining the orientation of the second harmonic-active moiety    with respect to the species;-   (c) measuring the polarization of second-harmonic light to determine    the orientation of the second harmonic-active moiety with respect to    the planar surface; and-   (d) determining the orientation of the species within the planar    surface from the orientation of the moiety with respect to the    surface as determined in step (c) and from the orientation of the    moiety with respect to the species as determined in step (b).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Absorption spectra of the SH-active moiety (dye)(oxazolederivative), cytochrome (cyt) c and dye-cyt c (amine) conjugate. TheSH-active moiety is covalently attached to amine groups on the protein'ssurface and, from the absorbance and known extinction coefficients, isbound at a mole ratio of 1.5:1 (dye:protein) in the conjugate.

FIG. 2. The SH intensity spectrum of the cyt c-amine conjugate (1.5:1dye:protein mole ratio; solid line) and cyt c-cysteine conjugate (0.4:1dye:protein mole ratio; ♦♦♦ line) at the air-water interface at bulkconcentrations of about 15 μM. The fundamental wavelength was set to 804nm and the spectrum displays the characteristic 2ω peak. The tail of thetwo-photon fluorescence is visible in the spectrum of the amineconjugate. I_(SH) (cps) is the intensity of SH light in counts persecond.

FIG. 3. Adsorption isotherm of the cysteine-cyt c conjugate (SHintensity vs. bulk [conjugate]) with error bars. The curve was fitted toa Langmuir adsorption model and, from this, a free energy of adsorptionof ΔG=−11 kcal/mole and the number of absorbates at the interface weredetermined.

FIG. 4. SH intensity spectrum of the oxazole dye alone at the air-waterinterface. The bulk dye concentration is about 700 μM. Although the peakSH intensity is comparable to that for the cysteine conjugate, the bulkconcentration of dye is at a factor of about 40 higher than that foreither the cysteine or the amine conjugates. Accordingly, the (twophoton) fluorescence background is much higher.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are presented as an aid in understanding thisinvention.

As used herein second harmonic refers to a frequency of light that istwice the frequency of a fundamental beam of light. A secondharmonic-active moiety is a substance which when irradiated with afundamental beam of light generates a second harmonic of thefundamental.

Having due regard to the preceding definitions, the present inventionconcerns a method for detecting a molecule at an interface, whichcomprises labeling the molecule with a second harmonic-active moiety anddetecting the labeled molecule at the interface using a surfaceselective technique.

In different embodiments of the invention, the surface selectivetechnique is second harmonic generation or sum-frequency generation. Sumfrequency generation (SFG) is a nonlinear, optical technique wherebylight at one frequency (ω₁) is mixed with light at another frequency(ω₂) to yield a response at the sum frequency (ω₁+ω₂) (Shen, 1984,1989). SFG is particularly useful for the detection of molecules atsurfaces through their characteristic vibrational transitions and, inthis case, is essentially a surface-selective infrared spectroscopy withω₁ and ω₂ at visible and infrared frequencies.

In different embodiments of the invention, the molecule to be detectedis a protein, a nucleic acid, a lipid, or a carbohydrate. In differentembodiments, the nucleic acid is a ribonucleic acid (RNA) or adeoxyribonucleic acid (DNA). In different embodiments, the DNA isgenomic DNA or cDNA.

In different embodiments, the molecule to be detected is a pollutant orother environmentally important molecule.

In different embodiments, the molecule is on a surface of a nanoparticleor a polymer bead.

In different embodiments, the second harmonic-active moiety is bound tothe molecule by a specific interaction or by a non-specific interaction.In different embodiments, the specific interaction comprises a covalentbond or a hydrogen bond. In different embodiments, the secondharmonic-active moiety is specific for an amine group or for asulfhydryl group on the molecule to be detected. In one embodiment, thenon-specific interaction comprises an electrostatic interaction.

In one embodiment, the second harmonic-active moiety comprises aplurality of individual second harmonic-active labels which each have anonlinear susceptibility and are bound together in a fixed anddeterminate orientation with respect to each other so as to increase theoverall nonlinear susceptibility of the second harmonic-active moiety.

In different embodiments, the interface is at a membrane, a liposome, acell surface, a viral surface, a bacterial surface, or a biosensor. Indifferent embodiments, the interface is a vapor-liquid interface, aliquid-liquid interface, a liquid-solid, or a solid-solid interface. Inone embodiment, the vapor-liquid interface is an air-water interface. Inone embodiment, the liquid-liquid interface is an oil-water interface.In different embodiments, the liquid-solid interface is a water-glassinterface or a benzene-SiO₂ interface.

The present invention provides for the use of any of the methodsdescribed herein to detect binding of a protein to a receptor on amembrane. The invention also provides for the use of any of the methodsdescribed herein to detect binding of a virus to a cell. The inventionfurther provides for the use of any of the methods described herein tostudy protein-protein interaction at an interface or to study cell-cellinteraction.

The invention provides a method for detecting a molecule in a medium,which comprises:

-   (a) labeling a surface with a second harmonic-active moiety wherein    the second harmonic-active moiety specifically interacts with the    molecule to be detected,-   (b) exposing the surface to the medium thereby creating an interface    at the surface,-   (c) detecting the second harmonic-active moiety at the interface by    measuring a signal generated using a surface selective technique,    and-   (d) detecting a change in the signal when the molecule interacts    with the second harmonic-active moiety, thereby detecting the    molecule in the medium.

In different embodiments of the method, the surface is on a nanoparticleor a polymer bead. In different embodiments, the surface selectivetechnique is second harmonic generation or sum-frequency generation.

In different embodiments, the molecule to be detected is a pollutant ora charged species. In different embodiments, the pollutant is lead orpolychlorinated biphenyl. In one embodiment, the charged species is achloride ion.

In one embodiments, the interaction between the second harmonic-activemoiety and the molecule to be detected is an antibody-antigeninteraction.

In different embodiments, the medium contains an amount of the moleculeto be detected, the change in the signal when the molecule interactswith the second harmonic-active moiety is a quantitative change, and theamount of the molecule in the medium can be determined from the changein the signal.

This invention provides a method for determining the orientation of amolecular species within a planar surface, which comprises:

-   (a) labeling the species with a second harmonic-active moiety which    specifically binds to the species;-   (b) determining the orientation of the second harmonic-active moiety    with respect to the species;-   (c) measuring the polarization of second-harmonic light to determine    the orientation of the second harmonic-active moiety with respect to    the planar surface; and-   (d) determining the orientation of the species within the planar    surface from the orientation of the moiety with respect to the    surface as determined in step (c) and from the orientation of the    moiety with respect to the species as determined in step (b).

In one embodiment of the method, the orientation of the secondharmonic-active moiety with respect to the species is determined usingx-ray crystalography. In different embodiments, the planar surface isselected from the group consisting of an organic material surface, aninorganic material surface, a polymeric material surface, a mineralsurface, a clay surface, a biological membrane surface, and a syntheticmembrane surface. In different embodiments, the molecular species isselected from the group consisting of an organic species, an inorganicspecies, a polymeric species, a protein, a lipid, a nucleic acid, and acarbohydrate.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

The attachment of a second harmonic-active moiety to a molecule fordetection of the molecule at an interface can be illustrated by studyingSHG-labeled cytochrome c at the air-water interface. Since the x-raycrystal structure of cytochrome c has been solved to atomic resolution,experiments can be designed to randomly or selectively label variousamino acid side-chains on the protein's surface. For instance, becauseonly one surface cysteine exists on cytochrome c, SHG can be used todetect the orientation of the cysteine conjugate at the air-waterinterface through a null angle technique. The free energy of adsorptionof the SH-active moiety-protein conjugate to the air-water interface canalso be measured.

Theoretical Considerations

The production of second harmonic light from an interface can bedescribed by the following equation: $\begin{matrix}{{I\left( {2\omega} \right)} = {\frac{32\pi^{3}\omega^{2}\sec^{2}\Theta}{c^{3}{ɛ(\omega)}{ɛ^{1/2}\left( {2\omega} \right)}}{{{{\overset{\rightarrow}{e}\left( {2\omega} \right)} \cdot \chi^{(2)}}\text{:}{\overset{\rightarrow}{e}(\omega)}{\overset{\rightarrow}{e}(\omega)}}}^{2}{I^{2}(\omega)}}} & (1)\end{matrix}$where I(2ω) and I(ω) are the intensity of the second harmonic andfundamental light, respectively, χ⁽²⁾ is the second-order nonlinearsusceptibility tensor, ē(ω) and ē(2ω) the products of the Fresnelfactors and the polarization vectors for the light beams, c is the speedof light, ε is the index of refraction, and Θ is the angle between thereflected harmonic light and the surface plane (Heinz, 1991). Thesurface nonlinear susceptibility χ⁽²⁾, neglecting local-field effects,isχ⁽²⁾=N_(s)<α⁽²⁾>  (2)where N_(s) is the total number of molecules per unit area at theinterface and <α⁽²⁾> is the average over the orientational distributionof the nonlinear polarizabilities in these molecules. Equation (2) canbe more explicitly expressed asχ_(ijk) ⁽²⁾=N_(s)<T_(iλ)T_(jμ)T_(kυ)>α_(λμυ) ⁽²⁾  (3)following Reider and Heinz (1995), where α⁽²⁾ _(i′j′k′) refers to themolecular nonlinear polarizability in the coordinate system of themolecule, T_(iλ) is the transformation tensor which relates thelaboratory and molecular frames of reference, and the average is takenover the orientational distribution of the molecules at the interface.From equations 1 and 2, the intensity of second harmonic radiation isquadratic with the surface density N_(s) of aligned molecules.

In the use of SHG-labels for molecules (nonlinear polarizability α_(L)⁽²⁾ per label), the distribution of the labels will determine theorientational average of the labels' polarizability and therefore the SHintensity. Typically, the labels will not be oriented in the samedirection, and so the SH intensity will be reduced; in the limit of manyrandomly distributed labels, the SH intensity will approach zero.Labeling ratios can therefore be adjusted according to the particularmolecule under study to maximize the net SH signal from several labels.One could also use ‘super-labels’, in which a number of individuallabels are bound together in a fixed and determinate orientation withrespect to each other, in order to maximize the overallhyperpolarizability. The molecules, and therefore the labels, must alsoexhibit a net orientation at the interface to produce a nonlineareffect. This requirement could be an advantage, however, in detectingspecific interactions, for example with protein-receptor recognitionwhich leads to a net protein orientation. Furthermore, throughmutagenesis, one can specifically engineer proteins for the purpose ofplacing SHG-labels in pre-determined positions.

Methods

Amine- and sulfhydryl-specific dyes (Molecular Probes; Eugene, Oreg.),derivatives of an oxazole dye which has been shown to be highlySH-active, were covalently attached to cytochrome c via either a surfacelysine or cysteine amino acid, respectively. Cytochrome c (Horse heart,Sigma) is a soluble, globular protein of about 12 kDa molecular weightwhich participates in biological electron transfer reactions through itsheme cofactor. The protein has, from an examination of the crystalstructure (code 1HRC—Protein Databank; 1.9 Å resolution), 19surface-exposed lysine side-chain amino groups, the target of theacylating dye, and a single, surface sulfhydryl group (cysteine 17). Theprotein has a net charge of +9 at pH 7 and a maximum ground state dipolemoment of 60 Debye calculated by using a standard program and the Charmcharge set (Gunner et al., 1996 and references therein). Cytochrome cwas covalently labeled with either an amine- or sulfhydryl-selectivederivative of oxazole pyridinium, both of which carry a positive +1charge, and purified by extensive dialysis according to prescribedprocedure and previous work (Salafsky et al., 1996). Thesulfhydryl-specific dye was1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridiniumtrifluoromethanesulfonate (PyMPO epoxide), and the amine-specific dyewas1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumbromide (PyMPO, SE). Non-covalently bound dye could be completelyremoved by successive dialysis steps as established by a controlexperiment with a non-reactive oxazole dye. The degree of labeling couldbe determined by measuring the absorption spectrum of the protein-dyeconjugate. Dye derivatives were obtained from Molecular Probes, and theparent compound has been shown to be highly SH-active at neutral pH(Salafsky and Eisenthal, 2000), the latter being of importance forbiological studies. Protein was reacted with the dye at a 5:1 mole ratio(dye:protein) in distilled water (sulfhydryl-specific reaction) orsodium bicarbonate at pH 8.3 (amine-specific).

The second harmonic generation set-up has been described previously indetail (Eisenthal, 1996; and references therein). Briefly, the beam ofan argon ion laser (10.5 W) is directed into the cavity of a titaniumsapphire mode-locked laser (Tsunami). The output, at a repetition rateof 82 MHz with ˜150 fs pulse duration (800-834 nm adjustable), was usedas the fundamental light. The fundamental light was directed on to thereflecting plane of the water surface (double distilled water in aTeflon dish) at an angle of 70° to the surface normal, and was removedafter the sample by a filter. Although the two-photon fluorescence ofthe dye is easily visible as a greenish-yellow glow, it is spectrallywell-separated from the 2ω wavelength due to a Stokes shift of ˜130 nm.In addition, the reflection set-up collects only a small portion of thelight emitted, which further reduces the amount of detectedfluorescence. The second harmonic light was collected and focused into amonochromator. For all experiments, the polarization of the fundamentalwas set to 45° from the normal to the laser table. Detection wasaccomplished using a photomultiplier tube in single photon-countingmode.

Results and Interpretation

The intensity of SH light generated from the air-water interface at the2% peak was measured to be 50±12 counts per second. Cytochrome c hasbeen shown previously to adsorb to the air-water interface (Kozarac etal., 1987). When cytochrome c (without dye label) was added to the waterphase, even at a 5-10 mM concentration, the SH intensity from theinterface either remained unchanged or diminished slightly,demonstrating that the heme cofactor does not contribute significantlyto the SH signal through a χ⁽²⁾ process, as expected since the hememacrocycle is highly centrosymmetric. This finding is also consistentwith previous work in which the fundamental wavelength was tuned acrossthe heme absorption without effect on the SH signal.

A typical absorption spectrum of the dye-protein conjugate is shown inFIG. 1, along with spectra of the protein and dye separately. In FIG. 1,cytochrome (cyt) c is conjugated to an amine-reactive oxazole derivativeand the degree of labeling is about 1.5:1 mole (dye:cyt) using the knownextinction coefficients of the protein's heme group and the dye (9.5×10⁴M⁻¹ cm⁻¹ at 412 nm and 2.6×10⁵ M⁻¹ cm⁻¹ at 415 nm, respectively). At aconcentration of 15 μM in the subphase, the amine-labeled protein at theair-water interface produces a 2ω signal of order 10⁴ cps (FIG. 2). Thetail of the dye fluorescence is visible at about 480 nm and considerablysmaller in magnitude than the SH peak. The SH signal of thecysteine-labeled cytochrome c was also measured (FIG. 2) and exhibitsthe same 2ω peak as that of the amine conjugate, although the signal issmaller, around 10³ cps; this is expected given the smaller dye:proteinlabeling ratio (0.4:1 indicating about one dye every other protein) asmeasured from the conjugate's absorption spectrum.

The orientation of the dye molecule in the cysteine conjugate wasmeasured using a null angle technique (Salafsky and Eisenthal, 2000;Hicks, 1986). The SH light produced by this conjugate could becompletely nulled by setting the analyzer at an angle of 30° from thesurface normal, corresponding to a dye molecular orientation of 52°degrees from the normal under the assumption of a singlehyperpolarizability element and a delta function in dye distribution.Because the dye orientation is measured with respect to the interfacialplane, an assumption of the dye's orientation on the surface of theprotein must be made to determine the protein's orientation in the labframe. If we assume that the dye is oriented so that its principal bondaxis (and likely SH-active axis) lies normal to the protein's surface,from an examination of the x-ray crystal structure the cytochrome c isoriented so that the heme plane is about 30° degrees from the normal tothe air-water interface, since the cysteine sulfhydryl group lies nearlyin the plane of the heme. An adsorption isotherm of the cysteineconjugate was also measured at the air-water interface by adding smallaliquots of the conjugate to the water subphase (FIG. 3). By fitting thedata to a Langmuir isotherm (Eisenthal, 1996), the free energy of theconjugate's adsorption to the air-water interface could be determined tobe ΔG=−11 kcal/mole which indicates a strong surface activity. However,when the oxazole dye alone (+1 charge) was added to the water phase atthe concentrations used for the conjugate (1-15 μM), it produced nodetectable change in the background SH signal. Only at much higherconcentrations of the dye (>0.5 mM) was the SH signal detectable, withan intensity of several thousand counts per second (FIG. 4). The freeenergy of the cytochrome adsorption to the air-water interface istherefore significantly larger than that for the free oxazole dye.

These results suggest the use of SHG-labels in other experiments. Forinstance, by designing an appropriate molecular platform at aninterface—a supported lipid bilayer system, for example—one might usethem to study protein-protein interactions at a membrane. Moreover,because SH light can be generated at non-planar surfaces (liposomes, forinstance, where their diameter is ˜λ, the wavelength of the fundamentallight; see Srivastava and Eisenthal, 1998), they may also find use instudies involving the surface of liposomes or biological cells.

The present application has demonstrated the concept of a ‘SHG-label’:the labeling of some molecule of interest with a SH-active moiety forstudying that molecule at an interface via a surface-selective techniquesuch as second harmonic generation. As an illustration of the technique,the protein cytochrome c was covalently labeled with amine- andsulfhydryl-specific SH-active dyes in order to study the dye-proteinconjugate at an air-water interface. Because of the SH-activity of thedye, the protein can be easily detected at the interface; if unlabeled,the protein is undetectable. Any protein of interest might thus bestudied at an interface using a SHG-label. The label's chemicalspecificity, its SH cross-section, or its absorption and resonancewavelength can be changed in accordance with the demands of a particularrequirement. SHG-labels for proteins should prove useful in studies ofprotein-receptor binding at interfaces of supported membranes, liposomesor cells. SHG-labeling should also prove useful for studies of othermolecules including nucleic acids, lipids, carbohydrates, nanoparticles,and polymer systems.

References

Eisenthal, K. B. 1996. Photochemistry and Photophysics of LiquidInterfaces by Second Harmonic Spectroscopy. J. Phys. Chem.100:12997-13006.

Gunner, M. R., Nicholls, A., Honig, B, J. 1996. Electrostatic Potentialsin Rhodopseudomonas Viridis Reaction Centers: Implications for theDriving Force and Directionality of Electron Transfer. J. Phys. Chem.100:4277-4291.

Heinz, T. F. 1991. Second Order Nonlinear Optical Effects at Surfacesand Interfaces. In Nonlinear Surface Electromagnetic Phenomena. Ponath,H. E. and Stegeman, G. I., editors. Elsevier/North Holland, Amsterdam.Chapter 5.

Hicks, J. M. 1986. Studies of Chemical Processes in Liquids Using ShortLaser Pulses: 1. The Dynamics of Photoisomerization of Polar Moleculesin Solution 2. Studies of Liquid Surfaces by Second Harmonic GenerationPh.D. dissertation, Columbia University.

Kozarac, Z., Dhathathreyan, A., Mobius, D. 1987. Interaction of Proteinswith Lipid Monolayers at the Air-Solution Interface Studied byReflection Spectroscopy. Eur. Biophys. J. 15:193-196.

Reider, G. A. and Heinz, T. F. 1995. Second-order Nonlinear OpticalEffects at Surfaces and Interfaces In Photonic Probes of Surfaces.Halevia, P., editor. Elsevier Science, Amsterdam. Chapter 9.

Salafsky, J. S. and Eisenthal, K. B. 2000a. Second HarmonicSpectroscopy: Detection and Orientation of Molecules at a BiomembraneInterface. Chem. Phys. Lett. 319:435-439.

Salafsky, J. S. and Eisenthal, K. B. 2000b. Protein adsorption atinterfaces detected by second harmonic generation. J. Phys. Chem. B.104: 7752-7755.

Salafsky, J. S., Groves, J. T., Boxer, S. G. 1996. Architecture andFunction of Membrane Proteins in Supported Lipid Bilayers: A Study withPhotosynthetic Reaction Centers, Biochemistry. 35:14773-14781.

Shen, Y. R. 1984. The Principles of Nonlinear Optics, John Wiley & Sons,New York.

Shen, Y. R. 1989. Surface properties probed by second-harmonic andsum-frequency generation. Nature 337: 519-525.

Srivastava, A., Eisenthal, K. B. 1998. Kinetics of Molecular TransportAcross a Liposome Bilayer, Chem. Phys. Lett., 292:345-351.

1. A method for detecting a molecule which is labeled with a secondharmonic-active label at an interface, which comprises: (a) contactingthe labeled molecule with the interface such that the label has a netorientation at the interface; (b) detecting light emitted from theinterface using a surface selective technique so as to detect thelabeled molecule in contact with the interface, wherein an unlabeledmolecule at the interface is undetectable using the surface selectivetechnique, and wherein the second harmonic-active label ishyperpolarizable.
 2. The method of claim 1, wherein the surfaceselective technique is second harmonic generation or sum-frequencygeneration.
 3. The method of claim 1, wherein the molecule is a protein,a nucleic acid, a lipid, or a carbohydrate.
 4. The method of claim 3,wherein the nucleic acid is a ribonucleic acid (RNA) or adeoxyribonucleic acid (DNA).
 5. The method of claim 1, wherein themolecule is a pollutant.
 6. The method of claim 1, wherein the moleculeis on a surface of a nanoparticle or a polymer bead.
 7. The method ofclaim 1, wherein the second harmonic-active label is bound to themolecule by a specific interaction or a non-specific interaction.
 8. Themethod of claim 7, wherein the specific interaction comprises a covalentbond or a hydrogen bond.
 9. The method of claim 7, wherein thenon-specific interaction comprises an electrostatic interaction.
 10. Themethod of claim 1, wherein the second harmonic-active label is specificfor an amine group or a sulfhydryl group on the molecule.
 11. The methodof claim 1, wherein the second harmonic-active label comprises aplurality of individual second harmonic-active moieties which each havea nonlinear hyperpolarizability and are bound together in a fixed anddeterminate orientation with respect to each other so as to increase theoverall nonlinear hyperpolarizability of the second harmonic-activelabel.
 12. The method of claim 1, wherein the interface is at amembrane, a liposome, a cell surface, a viral surface, a bacterialsurface, or a biosensor.
 13. The method of claim 1, wherein theinterface is a vapor-liquid interface, a liquid-liquid interface, aliquid-solid, or a solid-solid interface.
 14. The method of claim 13,wherein the vapor-liquid interface is an air-water interface.
 15. Themethod of claim 13, wherein the liquid-liquid interface is an oil-waterinterface.
 16. The method of claim 13, wherein the liquid-solidinterface is a water-glass interface or a benzene-SiO₂ interface. 17.The method of claim 1, wherein the molecule is a protein and theinterface is at a receptor on a membrane.
 18. The method of claim 1,wherein the molecule is on a viral surface and the interface is at acell surface.
 19. The method of claim 1, wherein the molecule is aprotein and the interface is at a protein.
 20. The method of claim 1,wherein the molecule is on a cell and the interface is at a cellsurface.
 21. A method for detecting a molecule in a medium, whichcomprises: (a) labeling a surface with a first molecule which is labeledwith a second harmonic-active label, which is hyperpolarizable and at aninterface wherein the first molecule specifically interacts with asecond molecule to be detected, (b) contacting the surface with a mediumcomprising the second molecule, thereby creating an interface at thesurface such that the label has a net orientation at the interface. (c)detecting the first molecule at the interface by measuring a signalgenerated by the second harmonic-active label using a surface selectivetechnique, wherein an unlabeled molecule at the interface isundetectable using the surface selective technique, and (d) detecting achange in the signal when the second molecule interacts with the firstmolecule, thereby detecting the second molecule in the medium.
 22. Themethod of claim 21, wherein the surface is on a nanoparticle or apolymer bead.
 23. The method of claim 21, wherein the surface selectivetechnique is second harmonic generation or sum-frequency generation. 24.The method of claim 21, wherein the molecule is a pollutant or a chargedspecies.
 25. The method of claim 24, wherein the pollutant is lead orpolychlorinated biphenyl.
 26. The method of claim 24, wherein thecharged species is a chloride ion.
 27. The method of claim 21, whereinthe interaction between the second harmonic-active labeled molecule andthe molecule to be detected is an antibody-antigen interaction.
 28. Themethod of claim 21, wherein the medium contains an amount of themolecule to be detected, the change in the signal when the moleculeinteracts with the second harmonic-active labeled molecule is aquantitative change, and the amount of the molecule in the medium isdetermined from the change in the signal.
 29. The method of claim 21,wherein the molecule to be detected is labeled with a secondharmonic-active label.
 30. A method for detecting an interaction betweena first molecule which is labeled with a second harmonic-active labeland a second molecule, which comprises: (a) contacting the firstmolecule at an interface with a medium comprising the second moleculesuch that the label has a net orientation at the interface wherein thefirst molecule specifically interacts with the second molecule; and (b)detecting an interaction between the first molecule and the secondmolecule at the interface by measuring a signal generated by the secondharmonic-active label using a surface selective technique, wherein anunlabeled molecule at the interface is undetectable using the surfaceselective technique, and wherein the second harmonic-active label ishyperpolarizable.
 31. The method of claim 30, wherein said secondmolecule is labeled with a second harmonic-active label.
 32. The methodof claim 21, wherein the first molecule or the second molecule isselected from the group consisting of a lipid, carbohydrate, protein andnucleic acid.
 33. The method of claim 30, wherein the first molecule orthe second molecule is selected from the group consisting of a lipid,carbohydrate, protein and nucleic acid.
 34. The method of claim 1,wherein the interface is a cell surface and the labeled molecule isprepared outside of the cell.